Antigen presenting cells (APCs) are naturally occurring cells whose function is to present both "self" and "foreign" proteins (antigens) to the immune system. When antigens are effectively presented by APCs, they can activate T lymphocytes to recognize and fight infections as well as some types of cancer (Shimizu, J. et al. 1991 J. Immunol. 146:1708-1714; Zou, J. et al. 1992 Cancer Immunol. Immunother. 35:1-6; Takahashi, H. et al. 1993 International Immunology 5:849-857). Antigen-pulsed APCs have traditionally been prepared in one of two ways: (1) small peptide fragments, known as antigenic peptides, are "pulsed" directly onto the outside of the APCs (Mehta-Damani, A. et al. 1994 J. Immunol. 153:996-1003); or (2) APCs are incubated with whole proteins or protein particles which are then ingested by the APCs. These proteins are digested into small peptide fragments by the APC and eventually carried to and presented on the APC surface (Cohen, P. A. et al. 1994 Cancer Res. 54:1055-1058).
After APCs are prepared by one of the above methods, they can be injected back into a patient as a "vaccine," eventually reaching locations such as lymph nodes where they present the desired antigen to T lymphocytes (Inaba, K. et al. 1990 J. Exp. Med. 172:631-640 1990 [published erratum appears in J. Exp. Med. 1990 172(4):1275]). In another treatment, T lymphocytes are removed from a patient and stimulated to grow in culture by contact with the APCs (Cohen, P. A. et al. 1993 J. Immunother. 14:242-252). This latter approach can be used to propagate large numbers of "antigen specific" T lymphocytes which can be given to the patient as "adoptive immunotherapy."
An effective APC has several important properties: (1) it retains the peptide antigen on its cell surface long enough to present it to T lymphocytes; (2) it should process (ingest and digest) whole proteins or particles into peptide fragments as described above; (3) it can be activated to express additional "costimulatory" and adhesion molecules on its surface membrane which help T lymphocytes respond appropriately after encountering antigen on the APC surface.
Because effective antigen presentation requires a complicated system of cellular signals, researchers have concentrated on collecting human cells whose primary natural function is antigen processing and presentation. While a wide variety of cell types such as monocytes, macrophages, B cells and dendritic cells have a demonstrated ability to present antigen, extensive evidence indicates that the dendritic cell (DC) is nature's most potent antigen-presenting cell. DCs can express all of the necessary costimulatory and presentation molecules with great flexibility. In addition, dendritic cells' only known function is antigen presentation. While other types of APCs are capable of resensitizing T lymphocytes to previously encountered antigens (so-called "recall" antigens), DCs are thought to be most responsible primary sensitization of T lymphocytes (Croft, M. et al. 1994 J. Immunol. 152:2675-2685).
DCs are derived from "myeloid precursor" cells in the bone marrow which also give rise to monocytes and macrophages (Thomas, R. et al. 1994 J. Immunol. 153:4016-4028). It is also possible that monocytes are themselves the immediate precursors of both DCs and macrophages. As support for this theory, researchers have found that monocytes are capable of developing into cells morphologically and immunophenotypically identical to either DCs or macrophages in culture. This finding indicates that lymphocytes which share the same bone marrow precursor are relatively uncommitted to a particular differentiation pathway for at least some portion of their development (Peters, J. H. et al. 1991 Pathobiology 59:122-126).
Because DCs are derived from the bone marrow, they must travel through the blood until they reach their destination organs. These target organs include virtually every organ in the body. Due to this essential transit through the blood, the blood itself is the richest available source of DCs in the human body. It has been estimated that 1-3% of all mononuclear blood cells are precommitted DCs (Thomas, R. et al. 1993 J. Immunol. 151:6840-6852). The 10-15% of peripheral blood mononuclear cells which are monocytes may also, at least in part, have the potential to differentiate into DCs (Peters, J. H. et al. 1991 Pathobiology 59:122-126).
A number of strategies have been developed by others to isolate and purify human DCs from peripheral blood. The two fundamental approaches involve (1) isolating bone marrow precursor cells (CD34.sup.+) from blood and stimulating them to differentiate into DCs; or (2) collecting the precommitted DCs from peripheral blood. While the first approach is of great theoretic interest, the patient must unadvantageously be treated with cytokines such as GM-CSF to boost the number of circulating CD34.sup.+ stem cells in the peripheral blood. Moreover, the procedures necessary to generate large numbers of DCs are costly and lengthy, and the function of DCs obtained in this fashion has not yet been proved adequate for many applications (Romani, N. et al. 1994 J. Exp. Med. 180:83-93). In addition, exposing DCs in culture to foreign proteins such as fetal calf serum can cause them to preferentially present these unwanted antigens.
The second approach for isolating DCs is to collect the relatively large numbers of precommitted DCs already circulating in the blood. Previous techniques for preparing mature DCs from human peripheral blood have involved combinations of physical procedures such as metrizamide gradients and adherence/nonadherence steps (Freudenthal, P. S. et al. 1990 Proc. Natl. Acad. Sci. 87:7698-7702); Percoll gradient separations (Mehta-Damani, et al. 1994 J. Immunol. 153:996-1003); and fluorescence activated cell sorting techniques (Thomas, R. et al. 1993 J. Immunol. 151:6840-6852). All of these methods are uniformly plagued by small final DC yields, quality control problems and/or probable functional alterations of the DCs due to physical trauma and the extended period of time required to complete these procedures.
One technique for separating large numbers of cells from one another is known as countercurrent centrifugal elutriation (CCE). In this technique, cells are subject to simultaneous centrifugation and a washout stream of buffer which is constantly increasing in flow rate. The constantly increasing countercurrent flow of buffer leads to fractional cell separations that are largely based on cell size.
It was demonstrated over ten years ago that when human blood mononuclear cells were separated by countercurrent centrifugal elutriation (CCE) into two basic fractions, then called "lymphocyte fraction" and "monocyte fraction," that the "monocyte" fraction possessed the ability to present a recall antigen, tetanus toxoid, to the "lymphocyte" fraction (Esa, A. H. et al. 1986 Immunology 59:95-99). However, these investigators did not attempt to use elutriation to specifically isolate dendritic cells from the peripheral blood. Additionally, these investigators did not question whether the monocyte fraction could sensitize T lymphocytes to antigens never previously encountered ("primary in vitro sensitization").
In experiments performed between 1992 and 1994, we performed CCE in the "traditional" manner. As was known, CCE separates cells by their size. Cell fractions were taken from the elutriation rotor at specific buffer flow rates, while the rotor spins at a constant rate. During the procedure, the buffer is constantly increasing in flow rate. In these previous experiments, we elutriated cell fractions from the rotor at a constant centrifugal speed of 3000 rpm. The following fractions were isolated in the traditional manner.
a "140" fraction (traditional lymphocyte fraction) was collected and used as a source of lymphocytes. This fraction was elutriated at a buffer flow rate of 140 cc/min. PA1 a "150" fraction, known as "intermediate" was discarded as is traditionally customary. This fraction was elutriated at a buffer flow rate of 150 cc/min. PA1 a "rotor off (R/0)" fraction (traditional "monocyte" fraction) was collected and used as a source of APCs. This fraction was collected by eluting the cells remaining in the initial sample after the rotor has stopped. PA1 a) providing a white blood cell fraction obtained from a mammalian source; PA1 b) separating the white blood cell fraction into four or more subfractions by countercurrent centrifugal elutriation; PA1 c) identifying the dendritic cell-enriched fraction from step (b); and PA1 d) collecting the enriched fraction of step (c). PA1 a) providing a white blood cell fraction obtained from a mammalian source; PA1 b) separating the white blood cell fraction of step (a) into four or more subfractions by countercurrent centrifugal elutriation; PA1 c) stimulating conversion of monocytes in one or more fractions from step (b) to dendritic cells by contacting the cells with calcium ionophore; PA1 d) identifying the denaritic cell-enriched fraction from step (c); and PA1 e) collecting the enriched fraction of step (d). PA1 a) providing a white blood cell fraction obtained from said mammal; PA1 b) separating the white blood cell fraction of step (a) into four or more subfractions by countercurrent centrifugal elutriation; PA1 c) stimulating conversion of monocytes in one or more fractions from step (b) to dendritic cells by contacting the cells with calcium ionophore; PA1 d) incubating said dendritic cells in the presence of an antigen specific for said disease-carrying agent; and PA1 e) reintroducing said dendritic cells into the mammal. PA1 While generally reliable for restimulating T-lymphocytes already sensitized to "recall" antigens such as tetanus, they were much less efficient in enabling primary sensitization to novel antigens, as well as to tumor antigens in general. PA1 FACS-guided separations indicated that whatever ability the monocyte fraction had to sensitize T lymphocytes to novel antigens was attributable to a small dendritic cell subpopulation. PA1 The monocytes in the monocyte fraction consistently failed to upregulate important costimulatory molecules such as B7.1 when they were cultured in the presence of a variety of test antigens. In contrast, the elutriated dendritic cell subpopulation did upregulate costimulatory molecules in the presence of certain antigens, and such upregulation was accompanied by improved antigen presentation. However, upregulation of costimulatory molecules in the dendritic cell subpopulation did not occur in the presence of most tested antigens, including tumor antigens. PA1 Color #1: CD3 alone, CD14 alone, etc; Leu M7 or Leu M9; anti-Class I, etc PA1 Color #2: HLA-Dq, B7.1, B7.2, CD25 (IL2r), ICAM, LFA-3, etc. PA1 to enable optimal processing of added proteins or uptake of pulsed peptides, PA1 optimal and controlled upregulation of essential costimulatory and presenting molecules on DCs such as B7.1, B7.2 and HLA-DR and HLA-DQ, PA1 conversion of the large monocyte population to an activated DC-like phenotype so that they also can participate in effective antigen processing and presentation.
Following elutriation, each fraction was cryopreserved in 10% DMSO so it could be stored and thawed for use at later times. However, as with prior attempts to isolate dendritic cells from other blood cells, this procedure did not provide good yields of dendritic cells. In a typical experiment, we only found a 2-3 fold enrichment of dendritic cells. Typically, we could obtain about 1.times.10.sup.7 dendritic cells from one isolation procedure.
Other cell isolation techniques have additionally used fluorescent activator cell sorting (FACS) to subselect DCs from other peripheral blood cells (Thomas, R. et al. 1994 J. Immunol. 153:4016-4028; Thomas, R. et al. 1993 J. Immunol. 151:6840-6852). However, all of these methods have inherent disadvantages. The final yields are relatively small, indicating losses of most of the initial DCs during inefficient and potentially traumatic purification processes.
Further, these methods are extremely time-consuming and prone to quality control problems. During these procedures purified DCs have been found to no longer be able to process antigen effectively after undergoing several days of mechanical purifications and/or FACS manipulations. In addition, monocytes, themselves a potential precursor of DCs, are lost in the purification process since they are part of the discarded 150 fraction. Mouse studies of dendritic cell isolation have demonstrated that enrichment methods which require several hours rather than days result in a more fully functional DC collection (Girolomoni, G. 1990 J. Immunol. 145:2820-2826).
Some investigators have shown rapid methods of isolating dendritic cells from mouse spleen cells by centrifugal elutriation followed by FACS analysis (Ossevoort, M. A. et al. 1992 J. Immunol. Methods 155(1):101-11). However, such rapid collection techniques have not been used to isolate dendritic cells from peripheral blood. In addition, a large number of the monocytes that co-migrate upon countercurrent centrifugal elutriation are wasted because there is no reliable method of isolating immunologically activated dendritic cells from monocytes. Therefore, a need exists for a method of isolating dendritic cells that results in large yields, but is quick and efficient. Further, a method of converting monocytes to dendritic cells needs to be developed to increase the yield of dendritic cells isolated from the peripheral blood.